Protruding from the membrane of the influenza A viruses are two proteins, the hemagglutinin (HA) and the neuraminidase (N). The hemagglutinins are subject to extreme variation through mutation and selection by the antibodies of the host animal. The hemagglutinins of influenza A viruses circulating in different animals fall into one of 16 evolutionarily related families called subtypes (called H1, H2 . . . H16). The hemagglutinins are further divided according to phylogeny between two related groups, Group 1 (H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16) and Group 2 (H3, H4, H7, H10, H14 and H15). The neuraminidase likewise falls into 9 families. Influenza A viruses are classified by the subtype of hemagglutinin (HA) and the neuraminidase (N) they exhibit. The influenza A viruses currently circulating in humans (H1N1 and H3N2) are respectively of the H1 and H3 subtypes of hemagglutinin and N1 and N2 subtypes of neuraminidase. The protective immune response to seasonal influenza viruses is dominated by isolate-specific, subtype-specific, neutralizing antibodies that bind strongly to the head of the HA, thereby blocking the function of the HA proteins in attaching the virus to the host-cell receptors (Wiley et al. 1987). “Antigenic drift”, the selection of strains of viruses with mutations on the surface of the HA head that decrease binding of neutralizing antibodies so that they do not protect against the new mutated (“drifted”) strain of virus, creates the regular need for updated seasonal influenza vaccines. The HA head of the novel 2009 (H1N1) pandemic influenza A virus of swine origin (nH1N1) was antigenically distinct (“antigenic shift”) from H1N1 seasonal influenza viruses that had been circulating in humans (Xu et al. 2010) and thus most humans lacked protective antibodies (Itoh et al. 2009).
The hemagglutinin protein mediates infectivity of the influenza virus, first binding the virion to the host cells, and second, fusing the membrane of the virus to the host cell membrane, enabling the viral genome to enter the cells (Wiley et al. 1987). The hemagglutinin protein has a globular head and a stem: the head of the hemagglutinin mediates the attachment of the virus to the host cells, whereas the stem of the hemagglutinin mediates the fusion of the membrane of the virus to the host cell membrane. Antibodies against the head of the hemagglutinin that only bind to the hemagglutinin of one strain or isolate of influenza virus (isolate/strain-specific) predominate in the usual human immune response to the seasonal influenza. If antibodies against the head of the hemagglutinin are of sufficient affinity/avidity and they sterically inhibit the receptor-binding site, they block infectivity of that strain/isolate of a sub-type of virus by inhibiting binding of the virus to the host cell.
Antibodies to the head of the hemagglutinin give protection from being infected twice by the same strain of influenza virus but these protective antibodies are very specific for a given isolate/strain of a subtype of influenza virus and thus only neutralize and protect against specific isolate/strains of influenza viruses (Wiley et al. 1987). As the replication of the influenza virus genome is very prone to errors, mutants of the virus readily arise. Those mutants that escape neutralization by antibodies against the head of the hemagglutinin of the original strain of virus tend to be selected to replicate because the new, mutant hemagglutinin has so low affinity for the protective antibodies against the original isolate/strain, that the antibodies can no longer neutralize the mutant virus. The mutant virus can then re-infect people immune to the original influenza virus giving rise to a new isolate of seasonal influenza. This process is called “antigenic drift” and explains why a new seasonal influenza vaccine, made up of the most dominant mutant strains of the circulating strains of influenza virus to induce neutralizing antibodies to the new mutant virus, is needed recurrently. Many mutations in the head of the hemagglutinin around the receptor-binding site that disrupt binding of neutralizing antibodies to the original isolate/strain of the virus are well-tolerated by the virus because they do not interfere with receptor binding.
There is another, more drastic change in the antigenicity of the hemagglutinin termed “antigenic shift”, that is characteristic of the viruses that cause influenza pandemics. The Spanish H1N1 influenza 1918 pandemic is estimated to have killed 50 million humans. Many subtypes of influenza virus circulate in animals, mainly aquatic birds, and infection of humans with these animal viruses can cause serious human infections, with highly pathogenic avian H5N1 influenza having a mortality of over 50% (Yen and Webster, 2009). Antigenic shift can occur when different strains of influenza, circulating in birds, swine and humans, infect the same host, enabling reassortment of genetic material, which is present on 8 pieces of RNA. Alternatively it can occur when viruses that are circulating in another species other than humans, infect humans and replicate in humans and transmit between humans. In the case of the 1918 “Spanish” H1N1 influenza pandemic, the influenza virus may have obtained all its 8 gene segments from avian species (Yen and Webster, 2009). In the case of the 1957 H2N2 influenza pandemic and the 1963 H3N2 influenza pandemic, reassortment between human influenza strains and H2 or H3 from avian species was involved (Yen and Webster, 2009). The 2009 pandemic influenza A H1N1 virus (pdmH1N1) was generated by reassortment between two swine influenza viruses, each with genes from avian, swine and human influenza viruses (Ding et al, 2009, Garten et al, 2009). Thus the HA of the 1918 H1N1, 1957 H2N2, 1968 H3N2 and 2009 pandemic influenza viruses ultimately originated from avian influenza. The HA of pdmH1N1 is distantly related to the 1918 pandemic influenza H1N1 virus (Xu et al, 2010). The 2009 pandemic influenza A H1N1 virus (pdmH1N1) was generated by reassortment between two swine influenza viruses, each with genes from avian, swine and human influenza viruses (Ding et al, 2009, Garten et al, 2009). The next pandemic may have genetic contributions from the highly pathogenic H5N1 avian influenza (H5N1) (Yen and Webster, 2009). Moreover, if the highly pathogenic H5N1 avian influenza (H5N1) undergoes genetic changes that make it readily transmissible in humans, it may become a pandemic.
The differences between the amino acid sequences of the ectodomain of the hemagglutinin of pandemic influenza viruses and the current circulating seasonal influenza viruses are substantial (“antigenic shift”). For example in the hemagglutinin of the 2009 pandemic H1N1 influenza, 21% of the amino-acid sequence of the ectodomain was non-identical with the corresponding sequence in seasonal H1N1 virus and ˜50% in the key epitopes on the HA head were non-identical (Xu et al, 2010). In addition, mutations in the hemagglutinin of human seasonal H1N1 influenza viruses (but not the HA of the 2009 pandemic H1N1 influenza virus that was derived from a swine H1N1 influenza virus) had introduced glycosylation sites in the head, blocking the access of neutralizing antibodies to the hemagglutinin head (Wei et al 2010a). The 2009 H1N1 virus spread so quickly it became a pandemic because the human population at that time, especially young people, had no circulating protective antibodies that could neutralize it (Itoh et al, 2009). Although there can be up to 20% amino acid differences in hemagglutinins within subtypes there can be 30-70% differences between the sequences of different subtypes of hemagglutinins (Karlsson Hedestam et al 2008). For example, the ectodomain of the hemagglutinin of an isolate of highly pathogenic avian influenza H5N1 exhibits ˜36% non-identical amino acids in the corresponding sequence in seasonal H1N1 influenza or the pandemic 2009 H1N1 influenza virus.
In contrast to mutations in the head of the hemagglutinin, mutations in the stem region of hemagglutinin are not well-tolerated because most mutations in the stem disrupt its structurally constrained role in mediating the fusion of the viral membrane to the host cell membrane, which is essential for infectivity. Thus different isolates and even subtypes of influenza virus exhibit little variation in the regions of the hemagglutinin stem that control fusion (Sui et al, 2009). Rare antibodies that bind to the hemagglutinin stem can neutralize influenza viral infectivity by inhibiting the conformational change in the stalk and thus the fusion of the viral membrane and the host cells membrane (Throsby et al, 2008, Sui et al, 2009, Ekiert et al, 2009). Because the stalk is conserved over many subtypes of influenza virus, the “heterosubtypic” antibodies that target the conserved sites of the hemagglutinin stem can neutralize multiple isolates and subtypes of influenza virus (Throsby et al, 2008, Sui et al, 2009, Ekiert et al, 2009).
However, for reasons that are not understood by those skilled in the art, antibodies against the hemagglutinin stem that can bind to the hemagglutinin stem of many isolates/strains and subtypes of influenza viruses (“cross-protective or “heterosubtypic” antibodies) are not induced at high frequency in normal infections or vaccinations with seasonal influenza. Corti et al. 2010) reported that heterosubtypic memory B cells were undetectable in normal humans but after seasonal influenza vaccination they could be detected in some individuals, although the frequency after seasonal influenza vaccination was very low and variable. They generated monoclonal antibodies from these rare heterosubtypic memory B cells and all but one bound to the hemagglutinin stem. However, the frequency of the heterosubtypic memory B cells after seasonal influenza vaccination was 26-200 fold less than that of memory B cells making antibodies specific for the seasonal influenza vaccine. The question of whether these heterosubtypic memory B cells actually gave rise to plasmablasts that secreted antibodies in response to seasonal influenza vaccine was not addressed in Corti et al (2010). Corti et al. (2010) did report that they detected, using a very sensitive assay, a small amount of heterosubtypic antibody in the serum that was insufficient to neutralize the H5N1 influenza virus. Corti et al. (2010) acknowledged that the magnitude of the response they saw was not useful for protection and finished their paper with “even in high responder individuals, heterosubtypic antibodies hardly reach effective neutralizing concentration in the serum.” Wrammert et al (2011) reported that they had generated monoclonal antibodies from newly generated blood plasmablasts shortly after seasonal influenza vaccination and found that none of the monoclonal antibodies were heterosubtypic antibodies targeted to the hemagglutinin stem.
The extremely low levels of cross-protective antibodies that bind with high affinity to the hemagglutinin of different isolates/strains and subtypes of virus (“cross-reactive or “heterosubtypic” antibodies) induced by infection (Wiley at al 1987) or vaccination (Corti et al, 2010) with seasonal influenza, explains why infection or vaccination with a given strain of seasonal influenza virus does not protect against other isolates/strains or subtypes of influenza virus. The lack of cross-protective and heterosubtypic antibodies is surprising given that most humans have been infected or vaccinated multiple times with different isolates/subtypes, with at least two subtypes of seasonal influenza (H1N1 and H3N2) and in some older individuals, also with the H2N2 virus.
Artificially engineered antibodies have been generated against the conserved region of the HA stem and they have been shown to neutralize multiple strains and subtypes of influenza (Throsby et al. 2008, Sui et al, 2009). They were generated by shuffling a library of human immunoglobulin heavy-chain and light-chain genes expressed in bacteriophages and selecting the resultant antibody fragments that bound to the H5 hemagglutinin of avian influenza (H5N1). These antibodies bound not only to H5 hemagglutinin but also to hemagglutinins from numerous other influenza subtypes, with the notable exception of H3 and H7 hemagglutinins from Group 2. Most of these artificial antibodies used one IGHV1-69 gene, and two studies showed by X-ray crystallography that the CDR1 and CDR2 encoded by this germline gene made the key contacts by these antibodies with the stem region of the H5 hemagglutinin (Ekiert et al. 2009; Sui et al. 2009). The light chain made minimal or no contacts with the hemagglutinin. This gene IGHV1-69 is expressed by most humans and therefore these heterosubtypic antibodies using IGHV1-69 would be expected to be made by most humans in large quantities given the recurrent antigenic stimulation with infections or vaccinations with seasonal H1N1 influenza. One group looked at the donor of the immunoglobulin gene library that yielded these artificially generated cross-reactive antibodies, and found that the donor did not have any circulating cross-reactive antibodies. Moreover Sui et al. (2009) concluded that such antibodies were not found amongst a large number of anti-influenza monoclonal antibodies cloned out of donors who had been vaccinated against seasonal influenza (Wrammert et al. 2008), and that some mechanism in the human immune system prevented these cross-reactive antibodies against the stem of hemagglutinin being produced in humans. There has been considerable interest in designing vaccines based on these observations (Chen et al. 2009). Corti et al. 2010 speculated that a new vaccine with an engineered immunogen that better exposed the stem region of the HA to achieve optimal presentation to B cells might result in heterosubtypic, cross-protective antibody responses. These attempts at producing a broad spectrum influenza vaccine have involved constructing artificial versions of the stem region of the hemagglutinin (Sagawa et al. 1996 and Steel et al. 2010) and have been based on the thesis that the stem of the hemagglutinin was masked by the bulky head domain, which was thus immunodominant (Steel et al. 2010; Wang et al 2010). However, although there was some protection induced by immunization with the “headless” HA, these researchers found no evidence (Sagawa et al. 1996) or very marginal evidence (Steel et al. 2010) that the protection was due to heterosubtypic neutralizing antibodies that could be transferred by serum from vaccinated mice. Another approach tried recently was to alter the presentation of the hemagglutinin by using DNA vaccination followed by a protein or viral vector boost (Wei et al, 2010b) which induced antibodies against the HA stem. However it was not shown by transfer of serum from vaccinated animals that the antibodies provided protection. These experiments were only done in naïve animals that had no previous experience of influenza vaccination or infection. The authors acknowledged that they might find different results, with human populations that had been previously exposed to influenza hemagglutinin.
Karlsson Hedestam et al (2008) and Kwong and Wilson (2009) drew a parallel with influenza virus and HIV-1 with respect that both viruses are extremely variable and both elicit isolate/strain-specific neutralizing antibodies against epitopes on peptide loops that both viruses can readily mutate and thus escape from the neutralizing antibodies induced by the original strain. In both diseases rare monoclonal antibodies have been generated that can neutralize a broad range of isolates and strains of viruses. Karlsson Hedestam et al (2008) and Kwong and Wilson (2009) pose the challenge to induce these broadly neutralizing antibodies by vaccination and contemplate new immunogens.
Antibodies are proteins that circulate in the bloodstream and have the ability to bind to and neutralize viruses and toxins and other pathogens. Antibodies come in billions of configurations and, given this structural diversity, it is likely that one or more of the antibodies in an individual will bind to any foreign substance or virus. The cells of the blood and immune system that make antibodies are termed “B cells”. Each B cell makes only one of the billions of different types of antibodies and has samples of that antibody displayed on its surface. When foreign substances (termed “antigens”), like the influenza virus, enter the body, they bind to those rare B cells that make a specific antibody that binds that foreign substance and stimulate those B cells to multiply. The multiplying B cells then undergo a process called “affinity maturation” in a structure called a germinal centre that develops in a lymph node. In this affinity maturation process, the genes encoding antibodies in the B cells accumulate somatic mutations. In the germinal centre, those B cells that make a mutated antibody that binds tightly to the antigen are selected. To undergo this “affinity maturation” process, B cells need the help of a related cell in the immune system called the “T cell” and the process of selection of the B cells that make the highest affinity antibodies is intimately involved with the mutual interactions between B cells and T cells (Allen et al, 2007, Victora, et al 2010, Schwickert et al 2011). Those B cells that make antibodies that bind to the hemagglutinin of an influenza virus can bind to the hemagglutinin or more likely to the virion and then internalize the protein or the virus (Russell et al, 1979). The B cells digest the hemagglutinin or virus and present small parts of the proteins (peptides) to the helper T cells. B cells need to present the peptide antigen to T cells in order to form a tight conjugate that will ensure that the T cell stimulates the B cell to proliferate and enter the germinal centre (Schwickert et al 2011) and then to proliferate and survive in the germinal centre (Allen et al, 2007, Victora, et al 2010).
The mechanism of this “affinity maturation” process involves two steps, first the induction of mutations in the antibody genes in B cells, and second, the survival of those B cells that make a more tightly binding (“higher-affinity”) antibody. The advantage that B cells that make the relatively higher affinity antibodies have over B cells that make lesser affinity antibodies results from them being better able to present peptide antigens to T helper cells. There is a limiting number of T cells that have receptors specific for the hemagglutinin or the proteins in the influenza virion and they form conjugates preferably with the B cells that make relatively higher affinity antibodies. These antigen-specific T cells become activated and stay in contact with the B cells while they induce them to proliferate and enter the germinal centres. When the B cells have entered the germinal centre, they need to present peptide antigens to the helper T cells to proliferate and survive. It has been recently established by elegant experiments that the relative affinity/avidity of the antibody expressed by a B cell determines its relative ability to compete with other B cells for presenting antigen to T helper cells (Victora et al 2010, Schwickert et al 2011). Thus B cells with higher affinity for the antigen will monopolize the T cell help.
The B cells that make the highest affinity antibodies and that thus survive the process of affinity maturation, become two types of specialized cells that leave the germinal centre and enter the blood, one specialized to secrete large amounts of antibodies (“antibody-secreting cells” or “plasma cells”), and one specialized to circulate in the blood for very long durations termed a “memory B cell”. If the memory B cell re-encounters the same foreign substance or antigen that induced it, it responds by rapidly producing antibodies specific to the antigen, making the individual “immune” to that antigen.
Memory B cells can live for decades in the body. Indeed circulating antibodies persisted in elderly humans for over 90 years after the 1918 pandemic. Similarly, neutralizing high-affinity monoclonal antibodies have been cloned from memory B cells binding to the head of the hemagglutinin of the 1918 pandemic H1N1 influenza virus from elderly humans, from people who were born before the pandemic meaning that the memory B cells had persisted for over 90 years (Yu et al, 2008). This exemplifies the longstanding, selective pressure that human influenza viruses are under by high-affinity, neutralizing antibodies against the hemagglutinin head. Moreover it indicates that memory B cells against the hemagglutinin head are a constant feature of the human immune system and their presence and specificity and affinity must be taken into account if it is contemplated to undertake influenza vaccination. If memory B cells re-encounter their specific antigen they can respond quickly in two ways. If they bind tightly to the antigen (ie the antibody the memory B cell makes is of high-affinity), they transform into a plasmablast, a cell specialized for secreting large amounts of their specific antibody (Paus et al 2006). If they bind more weakly to an antigen, for example a mutated version of the original antigen like an escape mutant of hemagglutinin from an “antigen-drifted” influenza virus, they re-enter the affinity-maturation process and undergo selection to bind more tightly to the new antigen (Paus et al 2006). Memory B cells that make antibodies that bind relatively weakly to the hemagglutinin of an influenza virus can still bind to the hemagglutinin or to the virion and ingest the protein or the viral proteins (Schwickert et al, 2011). Thus memory B cells that make antibodies that neutralize an original isolate of seasonal influenza but that do not neutralize a “drifted” isolate of seasonal influenza, can still readily bind hemagglutinin and present peptides from the hemagglutinin or an influenza virion to helper T cells. There are many shared T cell epitopes between different isolates and subtypes of influenza viruses (Doherty et al, 2008). Moreover T cell epitopes can come from all parts of the primary sequence of the protein, even the internal parts of the protein that are not displayed on the surface. Moreover if a B cell is making an antibody that binds to hemagglutinin, that B cell can bind and internalize a virion or a fragment of it. That B cell can then present to a T cell and get help from many T cells specific for individual peptides from the internal, conserved proteins of the virion (Russell et al, 1979).